One important aspect of regenerative medicine is the ability to introduce functional stem cells into patients to restore tissue function. This type of therapeutic approach will not be commonly used until several major potential problems have been addressed, including immune rejection and the risk of developing cancer.

Induced pluripotent stem cells (iPSCs) hold great promise in regenerative medicine: these cells are similar to embryonic stem cells (ESCs) but can be derived upon “reprogramming” of any mature cell type isolated from a patient. Thus, tissue-specific stem cells derived from iPSCs and re-injected into the same patient may not trigger immune rejection. However, before the full potential of iPSCs is achieved, we need to learn how to better generate these cells, control their maturation into tissue-specific stem cells and progenitors, and harness their tumorigenic potential.

Interestingly, ESCs and iPSCs share many characteristics of cancer cells, including their unlimited proliferation potential, and emerging evidence suggests that the mechanisms underlying the infinite proliferation of cancer cells and ESCs are intimately intertwined. Similarly, the progressions stages of tumorigenesis and cellular reprogramming to iPSCs share several characteristics, including changes in the packaging of the chromosomes.

Based on these observations, we propose to directly study the function of a major cancer pathway, the RB pathway, in cellular reprogramming and iPSCs. RB is a key tumor suppressor in humans. RB acts as a cellular brake that restricts cell division but has several other cellular functions, including in the control of cellular maturation. When RB is mutated, cells divide faster and become more immature, two features of cancer cells, but also of cells undergoing reprogramming.

We hypothesize that RB is an important regulator of cellular reprogramming and will test this idea using mouse and human cell types in culture. We believe that these experiments may identify novel and safer ways to induce the generation of iPSCs from adult cells or to improve existing protocols. Understanding the molecular details of the reprogramming process may lead to the development of small molecule compounds that can target precise proteins and/or transcription machinery involved in reprogramming. These experiments may also provide novel insights into the differences and similarities between tumor cells and iPSCs, providing new ways to suppress the tumorigenic potential of iPSCs and ESCs. Finally, better knowledge of the mode of action of RB family members may also allow for a better control of the differentiation of hESCs and iPSCs into tissue-specific stem cell populations.

Statement of Benefit to California:

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) hold the promise of treatments and cures for human diseases that affect millions of people. However, before these cells can be used in the clinic, a better understanding of the mechanisms controlling their proliferation and their capacity to produce a functional progeny is critically required. Our work on how the RB tumor suppressor gene may control the reprogramming of somatic cells into iPSCs may identify novel means to manipulate hESCs, to control the fate of these cells when transplanted into patients. Because hESCs have the capacity to form any type of cell in the human body, these experiments will be relevant to a large number of human diseases.

Despite significant decreases in the incidence and mortality rates of cancers in California over the past decade, nearly one out of every two Californians born today will still develop cancer at some point in their lives, and it is likely that one in five persons will die of the disease. Overall, in 2009, more than 50,000 people will die of cancer in California. These statistics underscore the need for the development of novel approaches to detect and treat human cancers. Given the similarities between tumor cells and embryonic cells, our work on the role of the RB tumor suppressor in hESCs and iPSCs may provide novel insights into the mode of action of RB in human cancer cells and may identify novel means to detect and treat cancer patients.

Thus, the proposed research may benefit a broad range of patients, from young children to senior citizens, in California and elsewhere.

Progress Report:

Induced pluripotent stem cells (iPSCs) hold great promise in regenerative medicine: these cells are similar to embryonic stem cells (ESCs) but can be derived upon “reprogramming” of any mature cell type isolated from a patient. Thus, tissue-specific stem cells derived from iPSCs and re-injected into the same patient may not trigger immune rejection. However, before the full potential of iPSCs is achieved, we need to learn how to better generate these cells, control their maturation into tissue-specific stem cells and progenitors, and harness their tumorigenic potential. Interestingly, ESCs and iPSCs share many characteristics of cancer cells, including their unlimited proliferation potential, and emerging evidence suggests that the mechanisms underlying the infinite proliferation of cancer cells and ESCs are intimately intertwined. Similarly, the progressions stages of tumorigenesis and cellular reprogramming to iPSCs share several characteristics, including changes in the packaging of the chromosomes.

Based on these observations, we proposed to directly study the function of a major cancer pathway, the RB pathway, in cellular reprogramming and iPSCs. RB is a key tumor suppressor in humans. RB acts as a cellular brake that restricts cell division but has several other cellular functions, including in the control of cellular maturation. When RB is mutated, cells divide faster and become more immature, two features of cancer cells, but also of cells undergoing reprogramming. We hypothesized that RB is an important regulator of cellular reprogramming and will test this idea using mouse and human cell types in culture. In the last year, we have performed experiments that largely support this hypothesis. We have found that, similar to its role in normal cell cycle, RB acts as a brake to normally restrict the reprogramming of cells into iPSCs. We have also found that RB is regulated in cells by enzymes that normally control the coating structure of chromosomes; these enzymes are thought to play a role in reprogramming, suggesting that RB may be a critical regulator of reprogramming by controlling the ability of reprogramming factors to modify the structure of the DNA. These experiments now provide a powerful system to analyze the molecular mechanisms underlying cellular reprogramming.

Our general goal is to better understand the differences and similarities between cancer cells and embryonic stem cells, to prevent tumor formation following stem cell transplantation but also to gain novel insights into the mechanisms of tumorigenesis and into the biology of embryonic stem cells. To this end, we have been studying how a tumor suppressor named Rb controls the dedifferentiation (or "reprogramming") of cells into induced pluripotent stem cells (iPS cells), which are similar to embryonic stem cells. (ES cells).

We have found that, similar to other tumor suppressors such as p53, Rb normally restricts the reprogramming process, both in human and mouse cells. We have also found that loss of RB does not change the proliferation rate of cells during reprogramming, suggesting that the enhanced efficiency of reprogramming observed in the absence of Rb is not due to a simple increase cell number. We are currently investigating the mechanisms by which Rb normally restricts the reprogramming process.

Our overarching goal is to understand the mechanisms controlling the balance between stem cell pluripotency, self-renewal, and tumorigenesis, to harness the full therapeutic promise of human embryonic stem cells (hESCs). To this end, we study the function of the RB gene family in stem cells. Our initial hypothesis was that RB family genes may control the reprogramming of somatic cells into iPSCs by interacting with chromatin remodeling factors to induce specific changes in the chromatin structure and control the expression of a specific program of genes. We found that loss of RB, but not of its family members p107 and p130, results in enhanced reprogramming of fibroblasts to iPS cells. In the past year, we have investigated this unique function of RB. In particular, we have performed high throughput RNA-seq and ChIP-seq experiments for RB early in the differentiation process to explore the mechanisms by which loss of RB may enhance reprogramming. We have also performed ChIP-Seq experiments with various chromatin marks to explore the relationship between RB loss and change sof the chromatin structure of cells early in reprogramming.

During the reporting period, we have pursued our work on the role of the retinoblastoma tumor suppressor during the reprogramming of mouse and human cells into induced pluriptoent stem cells (iPS cells). We have performed and analyzed genome-wide RNA-seq and ChIP-seq experiments to investigate how loss of RB promotes reprogramming. We have also tested candidate downstream mediators of RB in reprogramming using mouse genetics in vivo.